Method and device for producing optimized lipid-based micro/nano-bubbles

ABSTRACT

A method of producing lipid-based micro/nano bubbles includes steps of (a) preparing a lipid mixture including one or more first lipids with different phase transition temperature, and a second lipid bonding with a hydrophilic polymer moiety or molecules capable of getting across a lipid membrane and decreasing van der Waals forces between lipid bilayers; (b) emulsifying the lipid mixture with a solvent, to form a transparent lipid carrier solution; (c) placing the transparent lipid carrier solution in a closed vessel with halo-substituted hydrocarbon; (d) manipulating temperature of the transparent lipid carrier solution to be close to a main phase transition temperature thereof; and (e) agitating in a mechanical manner the vessel containing the transparent lipid carrier solution to form micro/nano bubbles within the closed vessel. This method contributes to form micro/nano bubbles with desired diameters in a way of optimal material utilization efficiency.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of Taiwan PatentApplication No. 102115967, filed on 2013 May 3, in the TaiwanIntellectual Property Office, the disclosure of which is incorporatedherein in its entirety by reference.

BACKGROUND OF THE DISCLOSURE Field of the Disclosure

The disclosure relates to lipid-based micro/nano-bubbles, and inparticular, to a method of preparing lipid-based micro/nano-bubbles witha controlled diameter in a way of an optimal material utilization withan optimal bubble concentration.

Related Art

Micro-bubbles are commonly applied to function as an ultrasound contrastagent in form of fine bubbles with diameter of 1-5 μm encapsulated by abiodegradable material. Such micro-bubble-based ultrasound contrastagent can circulate well in the blood stream and provide a significantecho-enhancement of perfusion in ultrasound imaging. Micro-bubbles havea high degree of echogenicity because of their much lower density(compared with human tissues) and compressible inner. Thesecharacteristics allow bubbles to reflect the ultrasound waves andfurther oscillate with these waves. The echogenicity difference betweenthe gas in the micro-bubbles and the soft tissue structures of the bodyis so significant that the ultrasound signal intensity may be enhancedby 20-40 dB. Thus, the micro-bubble-based ultrasound contrast agent canbe used to image blood perfusion in organs, measure blood flow rate inthe heart and other organs. Studies also show that micro-bubbles havethe similar characteristics to the contrast mediums of other medicalimaging systems for tumor diagnosis.

Another medical application for the micro-bubble-based ultrasoundcontrast agent is ultrasound molecular imaging. Through targetingligand-conjugated micro-bubbles to specific biomarkers on targettissues, ultrasound imaging can be used to highlight those tissues, suchas tumor, ischemia and inflammation areas, helping physicians achievingearly detections of various diseases. The similar application toultrasound molecular imaging is targeted therapy. Drug is encapsulatedin the micro-bubbles to be injected into the blood vessel. Once themicro-bubbles are accumulated remarkably in target tissues, drug releaseis actively triggered by focused ultrasound. Ultrasound causes bubbledestruction, which lowers the threshold for cavitation, resulting inmicro streaming and increased permeability of cell membranes. In otherwords, micro-bubbles serve as a vehicle to carry the anti-tumor drug andlocally release it when exposed to therapeutic ultrasound, which isreferred to as ultrasound-triggered drug release. Several studies alsoshowed the possibility of using micro/nano-bubbles to deliver genes intolive cells.

However, as currently used micro-bubbles are relatively bigger indiameter and unstable in blood circulation. Those micro-bubbles are hardto reach a sufficient accumulation in target tissue in such a limitedtime. Lack of a preparation method with optimal material utilizationefficiency (referred to lipid-to-bubble conversion ratio) also slowsdown the developments of functional micro-bubbles, such asligand-conjugated, drug-encapsulated and nanoparticle-loadedmicro-bubbles. Low material utilization efficiency results in a greatloss of those costly functional compounds that makes thecommercialization of functional micro-bubbles difficult. Therefore, itwould be greatly desirable to have micro-bubbles which can be wellcontrolled in diameter, stability, and material utilization efficiency(optimal material utilization efficiency).

SUMMARY OF THE DISCLOSURE

In view of the forgoing problems, the disclosure discloses a method ofproducing lipid-based micro/nano bubbles (including ultrasound contrastmicro-bubbles as well), in which the fluidity of the lipid membrane andthe main phase transition temperature of the transparent lipid carriersolution are determined by the composition of the lipid mixture, and theclosed vessel containing the lipid carrier is mechanically agitated atthe temperature around the main phase transition temperature of lipidcarrier, thus forming the micro-bubbles in a way of an optimal materialutilization efficiency. The size distribution and circulating stabilityof micro/nano bubbles can be improved through the fluidity regulation inthe same time.

In one aspect, this disclosure provides a method of producinglipid-based micro/nano bubbles. The method includes steps of (a)preparing a lipid mixture including one or more first lipids withdifferent phase transition temperature, and a second lipid bonding witha hydrophilic polymer moiety or molecules capable of getting across alipid membrane and decreasing van der Waals forces between lipidbilayers; wherein each of the first lipids includes a hydrophobic C8-C30end, and the second lipid is additionally bonding with a hydrophiliclong chain polymer moiety of molecular weight of 200-200,000; (b)emulsifying the lipid mixture with a solvent by mechanical means to forma transparent lipid carrier solution; (c) placing the transparent lipidcarrier solution in a closed vessel further with a predetermined gas orhydrophobic molecules; (d) manipulating temperature of the transparentlipid carrier solution to be close to the main phase transitiontemperature of the transparent lipid carrier solution; and (e) agitatingin a mechanical manner the closed vessel containing the transparentlipid carrier solution to form micro/nano bubbles within the closedvessel.

In another aspect, this disclosure provides a lipid mixture including:(a) one or more first lipids with different main phase transitiontemperatures, wherein each of the first lipids includes a C8-C30 alkylchain, the C8-C30 alkyl chain selected from the group consisting oflinear alkyl chain, alkenyl chain, alkylnyl chain, a fluoroalkyl chain,branched alkyl chain, and the combination thereof; and (b) a secondlipid bonding with a hydrophilic polymer moiety or molecules capable ofgetting across a lipid membrane and decreasing van der Waals forcesbetween lipid bilayers, wherein the second lipid includes alkyl chain asthat of the first lipids and is additionally bonding with a hydrophiliclong chain polymer moiety of molecular weight of 200-200,000; whereinthe lipid mixture is arranged such that the main phase transitiontemperature or the lipid membrane fluidity of a transparent lipidcarrier solution including the lipid mixture is capable to bemanipulated through manipulating the mixing ratio of the lipid mixture,so as to form micro/nano bubbles with desired diameters in a way ofoptimal material utilization efficiency by agitating the transparentlipid carrier solution under a temperature closed to the main phasetransition temperature of the lipid carrier in a mechanical manner.

In yet another aspect, this disclosure provides another lipid mixtureincluding: (a) one or more first lipids with different main phasetransition temperatures, wherein each of the first lipids includes aC8-C30 alkyl chain, the C8-C30 alkyl chain selected from the groupconsisting of linear alkyl chain, alkenyl chain, alkylnyl chain, afluoroalkyl chain, branched alkyl chain, and the combination thereof;(b) a second lipid bonding with a hydrophilic polymer moiety, whereinthe second lipid includes alkyl chain as that of the first lipids and isadditionally bonding with a hydrophilic long chain polymer moiety ofmolecular weight of 200-200,000; and (c) molecules capable of gettingacross a lipid membrane and decreasing van der Waals forces betweenbilayers; wherein the lipid mixture is arranged such that the lipidmembrane fluidity of a transparent lipid carrier solution including thelipid mixture is capable to be manipulated through manipulating themixing ratio of the molecules capable of getting across a lipid membranein the lipid mixture, so as to form micro/nano bubbles with desireddiameters in a way of optimal material utilization efficiency byagitating the transparent lipid carrier solution under a temperaturelower than the main phase transition temperature of the lipid mixture ina mechanical manner.

In further another aspect, this disclosure provides a device forproducing lipid-based micro/nano bubbles where a lipid mixture is usedas a starting material to be dissolved in distilled water, saline, orbuffered saline, the lipid mixture comprising one or more first lipidswith different main phase transition temperatures, and a second lipidbonding with a hydrophilic polymer moiety or molecules capable ofgetting across a lipid membrane and decreasing van der Waals forcesbetween lipid bilayers; wherein each of the first lipids includes aC8-C30 alkyl chain, the C8-C30 alkyl chain selected from the groupconsisting of linear alkyl chain, alkenyl chain, alkylnyl chain, afluoroalkyl chain, branched alkyl chain, and the combination thereof;and the second lipid includes alkyl chain as that of the first lipidsand is additionally bonding with a hydrophilic long chain polymer moietyof molecular weight of 200-200,000.

The above device includes: (a) a temperature controlling unit, formanipulating a temperature of the transparent lipid carrier solution tobe close to a main phase transition temperature of the transparent lipidcarrier solution; and (b) a mechanical agitator, for providingmechanical agitations on the transparent lipid carrier solution; whereinthe device is arranged such that the mechanical agitator is used toagitating a closed vessel containing the transparent lipid carriersolution having a temperature close to the main phase transitiontemperature to form the lipid-based micro/nano bubbles in the closedvessel.

This disclosure is advantageous particularly in that the cost formanufacturing the micro/nano bubbles can be reduced greatly bymanipulating the composition of the lipid mixture and mechanicallyagitating the vessel containing the lipid carrier at the temperatureclose to the main phase transition temperature of lipid carrier.Besides, the size or diameter of the micro/nano bubbles can also bedetermined as required by modifying the composition of the lipid mixtureand the temperature at which the lipid carrier is mechanically agitated.Once the composition is incorporated with high phase transitiontemperature materials, the circulating stability or the retention timeof encapsulated drug can also be improved. Therefore, this disclosuremay contribute significantly to applications of non-linear ultrasoundcontrast imaging, ultrasound molecular imaging and targeted therapywhere micro/nano-bubbles are involved. Further, a micro/nano bubbleagent with optimal concentration may show its usefulness in transdermaldelivery applications because of its fine characteristics in thecapabilities of smearing over the skin and oscillating with ultrasoundwaves to improve the permeability of cuticles. A optimized bubble agentfabricated by engineered lipid surfactants could be extensively appliedto cleaning applications such as cloths laundering, tooth washing orsemi-conductor washing processes.

The characteristics, realization and functions of the disclosure aredisclosed in the following description with reference to the preferredexemplified embodiments and the accompanying drawings

BRIEF DESCRIPTION OF THE DRAWINGS

This disclosure will become more fully understood from the detaileddescription given herein below for illustration only, and thus notlimitative of this disclosure, wherein:

FIG. 1 is the size distribution of DSPC-based micro/nano bubbles of thisdisclosure from Coulter counter particle analyzer (USP compliant);

FIG. 2 is a schematic view of a device for producing lipid-basedmicro/nano-bubbles according to a first embodiment of this disclosure;

FIG. 3 is a cross-sectional top view of the device of FIG. 2;

FIG. 4 is a schematic view of a mechanical agitator of this disclosure;

FIG. 5 is a schematic view showing the situation where the mechanicalagitator is incorporated with the device for producing lipid-basedmicro/nano bubbles;

FIG. 6 is a front view of a temperature controlling unit according toone embodiment of this disclosure;

FIG. 7 is a bottom view of the device for producing the lipid-basedmicro/nano bubbles of this disclosure; and

FIG. 8 is a front view of the device for producing the lipid-basedmicro/nano bubbles where the inlet airflow and outlet airflow areillustrated.

FIG. 9 is a schematic view showing the reciprocation motion of theclamp.

DETAILED DESCRIPTION OF THE DISCLOSURE

In the following description of preferred embodiments, reference is madeto the accompanying drawings which form a part hereof, and in which itis shown by way of illustration specific embodiments in which thedisclosure can be practiced. It is to be understood that otherembodiments can be used and structural changes can be made withoutdeparting from the scope of the embodiments of this disclosure.

Mechanical agitation is involved in a method of producing lipid-basedmicro/nano bubbles in this disclosure. A lipid mixture having a lipidmembrane is agitated to disrupt the original membrane structure,followed by reforming the membrane to encapsulate the introduced gas inthe innermost layer to form micro/nano bubbles. The membrane fluidity oflipid membranes indicates the ability of lipids moving on the membrane(refers to the ease of membrane disruption/reformation), which greatlyinfluences the micro/nano bubbles formed by the mechanical agitation inthroughput, size, and size distribution. In practice, the ultrasoundcontrast micro-bubbles above 8 μm in size will clog up lung capillaries.Therefore, it is important to control the size of the micro-bubblesproperly. However, it is difficult to manipulate both the size and sizedistribution of the ultrasound contrast micro-bubbles, so bubbles abovea predetermined diameter in size have to be removed finally as in priorart. Several clinical products which have around 2-5% micro-bubbleslarger than 10 μm have raised the concerns of safety issues in clinicaluses. In one embodiment, the method of producing ultrasound contrastmicro/nano bubbles includes the steps below.

As the initial step, a lipid mixture is prepared. Note that the lipidmixture could be prepared with a green manufacturing process which usesonly glycerol (propane-1,2,3-triol) or propylene glycol(propane-1,2-diol) as the initial solvent to mix and disperse all lipidmaterials (the solution temperature should be manipulated to thetemperature closed to the main phase transition temperature of the lipidmixture). Compared with prior arts, this clean manufacturing processavoids the use of toxic organic solvents such as methanol, toluene andchloroform.

The lipid mixture includes (a) one or more first lipids with differentmain phase transition temperatures and (b) a second lipid bonding with ahydrophilic moiety. Each of the first lipids includes a hydrophobicC8-C30 end. Alternatively, one or more categories of molecules capableof getting across a lipid membrane and decreasing van der Waals forcesbetween lipid bilayers may be added for regulating the main phasetransition temperature of the lipid mixture or further replaced for thesecond lipid. Besides, a solvent exclusive of water, i.e., non-watersolvent, may be also added to reduce the interactions between lipids forthe same purpose of regulating the main phase transition temperature ofthe lipid mixture. The hydrophobic C8-C30 ends may be, but not limitedto, a linear or branched alkyl chain, alkenyl chain, alkylnyl chain or afluoroalkyl chain or a combination thereof. Polymers with multiplehydrophobic C8-C30 ends could be also used as the first lipids.

Further, the first lipid may be, for example:

1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),

1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE),

1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DMPG),

1,2-dimyristoyl-sn-glycero-3-phosphoserin (DMPS),

1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA),

1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),

1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA),

2-dipalmitoyl-sn-glycero-3-phosphserine (DPPS),

1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG),

1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE),

1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),

1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DSPG),

1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),

1,2-distearoyl-sn-glycero-3-phosphate (DSPA),

1,2-distearoyl-sn-glycero-3-phosphserine (DSPS),

1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),

1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),

1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),

1,2-dioleoyl-sn-glycero-3-phosphate (DOPA),

1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG),

1,2-dioleoyl-sn-glycero-3-phosphserine (DOPS),

1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP),

1,2-distearoyl-3-trimethylammonium-propane (DSTAP),

dimethyldioctadecylammonium bromide (DMDDA),

1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaaceticacid (DPPE-DTPA),

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaaceticacid (DSPE-DTPA),

myristic acid, palmitic acid, stearic acid, oleic acid, tocopherols,tocotrienols, ascorbyl palmitate,

SPAN (Registered Trademark), Loxiol (Registered Trademark), Atlas™,Arlacel™, Emcol (Registered Trademark) or a combination thereof orderived polymers thereof.

Among others, combination of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine(DPPC) and 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DSPG)is used as the first lipid in this embodiment.

It should be understood that the main phase transition (solid to liquid)temperature of DPPC is 41 Celsius degree, while that of DSPG is 55Celsius degree. Lipid with higher phase transition temperature isadvantageous in reducing size (diameter) and increasing stability of theresultant micro/nano bubbles. The second lipid has a similar carbonchain structure (C8-C30, backbone) to the above first lipid and thesecond is additionally bonding with a hydrophilic long chain polymermoiety of molecular weight of 200-200000. The hydrophilic long chainpolymer moiety may be for example polyethylene glycol (PEG),polypropylene glycol, polyoxyethylene, polyvinylalcohol,polyvinylpyrrolidone and related copolymers, and peptide,deoxyribonucleic acid (DNA), or ribonucleic acid (RNA) or a combinationthereof.

The second lipid may be, but not limited to,

1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DPPE-PEG2000),

1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-3000] (DPPE-PEG3000),

1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-3000] (DPPE-PEG5000),

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000] (DSPE-PEG2000),

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000] (DSPE-PEG3000),

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000] (DSPE-PEG5000),

polyoxyethylene stearates, polyethylene glycol stearates, TWEEN(Registered Trademark), Myrj™, Atlas™, d-alpha-tocopheryl polyethyleneglycol 1000 succinate (Vitamin E TPGS), antibody-conjugated PEG-ylatedlipid, peptide-conjugated PEG-ylated lipid, DNA-conjugated PEG-ylatedlipid, RNA-conjugated PEG-ylated lipid, biotin-modified PEG-ylatedlipid, maleimide-modified PEG-ylated lipid, amine-modified PEG-ylatedlipid, and the combination thereof or the derived polymers thereof. Inthis embodiment, polyethylene glycol 40 stearates (PEG40S) are employed.

The molecule, which is able to get across the lipid membrane and todecrease van der Waals forces between lipid bilayers, may be but notlimited to, polyethylene glycol, peptide, albumin, amino acid, sugaralcohols, butane-1,3-diol, propane-1,2,3-triol, propane-1,2-diol,propane-1,3-diol, propan-1-ol, ethane-1,2-diol, ethanol, methanol anddimethyl sulfoxide, or a combination thereof. Propane-1,2,3-triol(glycerol) is used in this embodiment for improving the membranefluidity and ability of the lipid membrane to reconstruct itself.Particularly, some chemicals listed above like methanol and ethanol maygreatly increase the final throughput of the micro-bubbles under thetemperature far below the original main phase transition temperature(determined in water) of the lipid mixture even with a concentration ofthe range of 0.1 to 2 wt %.

As the second step, the lipid mixture including DSPG, DPPC, and PEG40Sblended with a solvent such as normal saline or buffered saline. Forexample the mixture of DSPG, DPPC, and PEG40S is blended with a solventof phosphate buffer saline containing 1 wt % of glycerol and a bilayerlipid membrane will be formed. In particular, since PEG40S functions toincreases the membrane fluidity of the lipid membrane, the bilayer lipidmembrane may be disrupted and reconstructed more easily during thefollowed agitation to form a monolayer structure, thereby encapsulatingthe introduced gas and increasing the final throughput of the micro/nanobubbles. The molecule capable of getting across the lipid membrane anddecreasing the van der Waals forces between lipid bilayers, i.e.,glycerol in this embodiment, functions similarly to PEG40S. Besides,DSPG with the higher main phase transition temperature in the firstlipid serves to decrease the membrane fluidity of the lipid membrane andstabilize the resultant micro-bubbles.

As the third step, the resultant solution formed by blending those lipidmaterials with the solvent is then mechanically emulsified until atransparent lipid carrier solution is formed. Sonication, high-speedagitation, high-pressure homogenization or membrane filtration may beemployed to mechanically emulsify the lipid mixture within the solvent.In this embodiment, a bath sonicator is used for sonication, and thetransparent lipid carrier solution is adjusted to have a concentrationof the lipid carrier of 3 mg/mL and a temperature of 20 Celsius degree.This step is directed to dispersing the lipid materials in thetransparent lipid carrier solution and decreasing the particle size ofthe lipid carrier, thus facilitating the subsequent preparation of themicro/nano bubbles. Note that the lipid concentration for fabricatingnano bubbles (bubbles with size less than 1 μm) should be typicallyhigher than 0.5 mg/mL. As the fourth step, the transparent lipid carriersolution used as the lipid carrier is placed in a closed vessel with aproper size. In this example, 1 mL of the transparent lipid carriersolution is added into the 1.8 mL closed vessel. Preferably, 0.5-1.0 mLof the transparent lipid carrier solution is used.

As the fifth step, the closed vessel is vacuumed and filled with apredetermined gas such as halo-substituted hydrocarbon(perfluorocarbon), inert gas, Sulfur hexafluoride, nitrogen, oxygen,air, or a combination thereof. The predetermined gas could also beintroduced into the vessel by sealing the vessel in a closed system orenvironment that contained with the predetermined gas orflushing/purging the vessel with the predetermined gas. Hydrophobicmolecules with specific functionalities could be further added into theclosed vessel in this step together with the predetermined gas. Thehydrophobic molecules may be, but not limited to, specific drug(s) to beincorporated into the lipid membrane of micro/nano bubbles for medicalapplications, such as ultrasound-triggered drug release,ultrasound-assisted tumor therapy and micro-bubble-based blood brainbarrier disruption. It will be appreciated that a person skilled in theart can understand the way for filling the predetermined gas and it willnot be addressed particularly here for avoiding unnecessary confusion.

As the sixth step, temperature of the transparent lipid carrier solutionis manipulated to be approximate to a main phase transition temperaturethereof by water bath for example, thus improving the membrane fluidity,ability of the lipid membrane to reconstruct itself, and the throughputof the micro-bubbles. For example, given the transparent lipid carriersolution with the main phase transition temperature of 46 Celsius degreecomposed of DPPC, DSPG, and PEG40S in a ratio of 1:1:1 (w/w/w), thereaction temperature of 43 Celsius degree at the next step will resultin a throughput of micro/nano bubbles three times higher than that of 20Celsius degree. In short, manipulating the temperature of thetransparent lipid carrier solution is favorable in preparing themicro/nano bubbles in a way having optimal material utilizationefficiency. However, it is noted that the temperature of the transparentlipid carrier solution may be manipulated to a temperature closed buthigher than its main phase transition temperature thereof, in case of adecreased temperature of the transparent lipid carrier solution duringthe subsequent agitating process in a mechanical manner. For example, ifthe main phase transition temperature of the transparent lipid carriersolution composed of DSPC, DSPE-PEG2000 in a ratio less than 10:4 (w/w)is 56 Celsius degree, since the temperature during the subsequentagitating process in a mechanical manner can only be maintained at about50 Celsius degree, the transparent lipid carrier solution is desirablyheated to 60 Celsius degree in advance for obtaining the high-throughputmicro/nano bubbles in a way having an optimal material utilizationefficiency (the throughput of the micro-/nano bubbles may be up to 4E+10bubbles/mL). Through the use of a high transition temperature material,so as DSPC, the average size could be reduced to the range of 400 to 700nm (depends on compositions).

FIG. 1 the size distribution of DSPC-based micro/nano bubbles of thisdisclosure from Coulter counter particle analyzer (USP compliant).

As the seventh step, the closed vessel containing the transparent lipidcarrier solution is agitated in a mechanical manner to form ultrasoundcontrast micro/nano bubbles within the closed vessel. The mechanicalagitation is realized by for example sonication, manual shaking,high-speed mechanical agitation, microfluidic device/T-focusing, orco-axial electrohydrodynamic atomization (CEHDA) micro-bubbling. Amechanical agitator is used at 4550 rpm for 45 seconds in thisembodiment, by which 99.9% of the resultant micro-bubbles have adiameter less than 8 μm. Preferably, the diameter is ranging from 0.2 μmto 8 μm. The way of high-speed mechanical agitation is favorable becausethe size and size distribution of the micro-bubbles can be controlledeffectively. Table I lists the average diameter (average from the rangeof 0.7 to 18 μm) of the micro-bubbles under different compositions ofthe transparent lipid carrier solution. The peak diameters of severallisted compositions may be much smaller than the average diametersdetermined from the measuring range of 0.7 to 18 μm.

TABLE I No. DPPC:DSPG:PEG40S (w/w/w) Average diameter (μm) 1 10:4:10.931 ± 0.007 2 10:4:2 1.083 ± 0.006 3 10:4:3 1.171 ± 0.114 4 14:0:21.814 ± 0.015 5 14:0:4 2.368 ± 0.061 6 14:0:6 2.861 ± 0.046

Alternatively, the lipid mixture includes (a) one or more first lipidswith different main phase transition temperatures, wherein each of thefirst lipids includes a C8-C30 alkyl chain;

(b) a second lipid bonding with a hydrophilic polymer moiety, whereinthe second lipid includes alkyl chain as that of the first lipids; and(c) at least one category of molecules capable of getting across a lipidmembrane and decreasing van der Waals forces between lipid bilayers. TheC8-C30 alkyl chain may be, but not limited to, a linear or branchedalkyl chain, alkenyl chain, alkylnyl chain, a fluoroalkyl group, or acombination thereof.

Polymers with multiple hydrophobic C8-C30 ends could be also used as thefirst lipids.

Further, the first lipid may be, for example,

1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),

1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE),

1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DMPG),

1,2-dimyristoyl-sn-glycero-3-phosphoserin (DMPS),

1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA),

1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),

1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA),

2-dipalmitoyl-sn-glycero-3-phosphserine (DPPS),

1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG),

1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE),

1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),

1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DSPG),

1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),

1,2-distearoyl-sn-glycero-3-phosphate (DSPA),

1,2-distearoyl-sn-glycero-3-phosphserine (DSPS),

1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),

1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),

1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),

1,2-dioleoyl-sn-glycero-3-phosphate (DOPA),

1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG),

1,2-dioleoyl-sn-glycero-3-phosphserine (DOPS),

1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP),

1,2-distearoyl-3-trimethylammonium-propane (DSTAP),

dimethyldioctadecylammonium bromide (DMDDA),

1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaaceticacid (DPPE-DTPA),

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaaceticacid (DSPE-DTPA),

myristic acid, palmitic acid, stearic acid, oleic acid, tocopherols,tocotrienols, ascorbyl palmitate, SPAN (Registered Trademark), Loxiol(Registered Trademark), Atlas™, Arlacel™, Emcol (Registered Trademark)or a combination thereof or derived polymers thereof.

The second lipid has a similar carbon chain structure (C8-C30, backbone)to the above first lipid and is bonding with a hydrophilic long chainpolymer moiety of molecular weight of 200-200,000. The hydrophilic longchain polymer moiety may be for example polyethylene glycol (PEG),polypropylene glycol, polyoxyethylene, polyvinylalcohol,polyvinylpyrrolidone and related copolymers, and peptide,deoxyribonucleic acid (DNA), or ribonucleic acid (RNA) or a combinationthereof.

The second lipid may be, but not limited to,

1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DPPE-PEG2000),

1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-3000] (DPPE-PEG3000),

1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-3000] (DPPE-PEG5000),

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000] (DSPE-PEG2000),

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000] (DSPE-PEG3000),

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000] (DSPE-PEG5000),

polyoxyethylene stearates, polyethylene glycol stearates, TWEEN(Registered Trademark), Myrj™, Atlas™, d-alpha-tocopheryl polyethyleneglycol 1000 succinate (Vitamin E TPGS), antibody-conjugated PEG-ylatedlipid, peptide-conjugated PEG-ylated lipid, DNA-conjugated PEG-ylatedlipid, RNA-conjugated PEG-ylated lipid, biotin-modified PEG-ylatedlipid, maleimide-modified PEG-ylated lipid, amine-modified PEG-ylatedlipid, and the combination thereof or the derived polymers thereof.

The molecule which is able to pass through the lipid membrane andfunctions to decrease van der Waals forces between lipid bilayers maybe, but not limited to, polyethylene glycol, peptide, albumin, aminoacid, sugar alcohols, butane-1,3-diol, propane-1,2,3-triol,propane-1,2-diol, propane-1,3-diol, propan-1-ol, ethane-1,2-diol,ethanol, methanol and dimethyl sulfoxide, or a combination thereof.

By manipulating the composition of the lipid mixture, the membranefluidity of the transparent lipid carrier solution and ability of thelipid membrane to reconstruct itself is capable to be manipulatedthrough manipulating the mixing ratio of the molecules capable ofgetting across a lipid membrane in the lipid mixture. Subsequently, thelipid carrier solution is subject to emulsion or mechanical agitation ata temperature lower than the main phase transition temperature of thelipid carrier to form the micro/nano bubbles with desired size of adiameter ranging from 0.2 to 8 μm (not limited to) in a way whereoptimal material utilization efficiency; wherein the lowered temperatureis a temperature ranging from 10 Celsius degree to 60 Celsius degree.

For obtaining the micro/nano bubbles in a way having optimal materialutilization efficiency at the temperature close to the main phasetransition temperature of the lipid carrier by using mechanicalagitation such as sonication, manual shaking, high-speed mechanicalagitation, microfluidic device/T-focusing, or co-axialelectrohydrodynamic atomization (CEHDA) micro-bubbling, the temperatureof the transparent lipid carrier solution may be increased in advance ofthe mechanical agitation by for example a water bath, dry heat ormechanical agitations themselves, and then be decreased to thetemperature around the main phase transition temperature of the lipidcarrier, followed by mechanically agitating the entire transparent lipidcarrier solution. Alternatively, for example, a heater/cooler system maybe combined with a mechanical agitator to facilitate the manipulation ofthe temperature of the lipid carrier.

FIG. 2 is a schematic view of a device 100 for producing lipid-basedmicro/nano-bubbles according to a first embodiment of this disclosure.

The lipid mixture as a raw material to be placed in the device 100includes (a) one or more first lipids with different main phasetransition temperatures, each having one or two alkyl chain with 8-30carbon atoms in length (C8-C30 alkyl chain) and (b) a second lipidbonding with a hydrophilic polymer moiety, or molecules capable ofgetting across a lipid membrane and decreasing van der Waals forcesbetween lipid bilayers. The second lipid has a similar carbon chainstructure (C8-C30, backbone) to the above first lipid, and the secondlipid is additionally bonding with a hydrophilic long chain polymermoiety of molecular weight of 200-200,000. The lipid mixture is used asa starting material to be dissolved in distilled water.

FIG. 3 is a cross-sectional top view of the device 100 of FIG. 2. Thedevice 100 includes a mechanical agitator 110 to generate mechanicalagitations and a temperature controlling unit 120 to manipulate thetemperature of the transparent lipid carrier solution to be closed themain phase transition temperature thereof.

If further agitations from the mechanical agitator 110 are acted on thelipid carrier placed that has been subject to preliminary agitations ina closed vessel 200 clasped by a clamp (not shown), micro/nano bubblesare thus formed therein.

FIG. 4 is a schematic view of the mechanical agitator 110 of thisdisclosure. The mechanical agitator 110 includes a clamp 111 serving toclasp the closed vessel 200, a lever 112 connected to and allowed to beagitated together with the clamp 111, a motor 113 connected with thelever 112 and providing integrally the clamp 111 and the lever 112 withpower for reciprocation, and a fastener (not shown) connected to an endof the lever 112 for restraining the movement thereof. The lever 112together with the clamp 111 may be reciprocated smoothly to achieveaforementioned mechanical agitations or emulsion when driven by themotor 113 with the restraint of the fastener. FIG. 5 schematically showsthe situation where the mechanical agitator 110 is incorporated with thedevice 100 for producing lipid-based micro/nano bubbles. In thisexample, the clamp 111 is disposed above the temperature controllingunit 120 to make the manipulation of the temperature controlling unit120 to the closed vessel 200 containing the lipid mixture and clasped bythe clamp 111 more convenient. In other words, even the clamp 111agitates in a reciprocating way horizontally (either linear orfigure-9-shaped as shown in FIG. 9) together with the lever 112 whendriven by the motor 113, the closed vessel 200 clasped by the clamp 111always stays overhead the temperature controlling unit 120 in positionregardless of the reciprocation. In this way, a satisfactory temperaturecontrolled performance can be obtained.

Further, as shown in FIGS. 2 and 3, the device 100 may include a casing130 for enclosing and accommodating all of the elements together withthe lipid mixture. A lid 131 may be provided to the casing 130 forfacilitating the take-in or take-out or replacement of the elements.FIG. 6 is a front view of the temperature controlling unit 120 accordingto one embodiment of this disclosure. The temperature controlling unit120 disposed within the casing 130 includes a first fan set 121, asecond fan set 122, a heating coil 123, and a sensor (not shown). As analternative, the temperature controlling unit 120 may be incorporated inthe clamp 111 or lever 112. FIG. 7 is a bottom view of the device 100for producing the lipid-based micro/nano bubbles of this disclosure. InFIG. 7, the temperature controlling unit 120 is located on the bottom ofthe casing 130 as can be observed therefrom.

The first fan set 121 is configured to generate an inlet airflow 310flowing towards the inside of the casing 130, while the second fan set122 is configured to generate an outlet airflow 320 flowing towards theoutside of the casing 130. The heating coil 123 is configured to heatthe inlet airflow 310, and the sensor is configured to detect thetemperature within the casing 130.

When the temperature within the casing 130 is to be increased to atarget temperature, the first fan set 121 and the heating coil 123 arestarted. However, the second fan set 122 may be optionally started toenhance the thermal convection under this condition. On the contrary,when the temperature within the casing 130 has to be decreased toanother target temperature, the second fan set 122 is started with thefirst fan set 121 optionally. FIG. 8 is a front view of the device 100for producing the lipid-based micro/nano bubbles of this disclosurewhere the inlet airflow 310 and outlet airflow 320 are illustrated forbetter understanding.

In another embodiment, the device 100 may include a dry heating piece(not shown) incorporated in the clamp 111 or lever 112. The heatingpiece helps the closed vessel 200 with a rapid temperature raise. Also,it is to be noted that the closed vessel 200 containing the transparentlipid carrier solution as the lipid carrier will possibly have a raisedtemperature due to a severe shaking imparted from the mechanicalagitator. Therefore, for example, if the raised temperature is higherthan the target temperature, the mechanical shaking may be stopped for awhile.

In this disclosure, the fluidity of the lipid membrane and the mainphase transition temperature of transparent lipid carrier solution aredetermined by the composition of the lipid mixture. The vessel 200containing the lipid carrier is mechanically agitated at the temperaturearound the main phase transition temperature of lipid carrier, thusforming the micro-bubbles in a way of optimal material utilizationefficiency. Hence, this disclosure is advantageous particularly in thatthe cost for manufacturing the micro/nano bubbles can be reducedgreatly.

In addition, size or diameter of the micro/nano bubbles can also bedetermined as required by the composition of the lipid mixture and thetemperature at which the lipid carrier is mechanically agitated.

The specific embodiments described above have been shown by way ofexample, and it should be understood that these embodiments may besusceptible to various modifications and alternative forms. It should befurther understood that the claims are not intended to be limited to theparticular forms disclosed, but rather to cover all modifications,equivalents, and alternatives falling within the spirit and scope ofthis disclosure.

1.-19. (canceled)
 20. A microbubble-containing composition prepared by a process comprising (a) preparing a lipid mixture consisting of a first lipid, or two or more first lipids with different phase transition temperature, a second lipid bonding with a hydrophilic polymer moiety and one or more molecules capable of getting across a lipid membrane and decreasing van der Waals forces between lipid bilayers; wherein each of the first lipids includes a hydrophobic C8-C30 end, and the hydrophilic polymer moiety has a long chain with molecular weight of 200-200,000; (b) emulsifying the lipid mixture with an aqueous solvent by mechanical means to form a transparent lipid carrier solution and then adjusting the transparent lipid carrier to 20 Celsius degree; (c) placing the transparent lipid carrier solution in a closed vessel comprising a predetermined gas or a hydrophobic molecule; and (d) agitating the closed vessel containing the transparent lipid carrier solution by sonication to form said microbubble-containing composition.
 21. The composition as claimed in claim 20, wherein the hydrophobic C8-C30 end is selected from the groups of linear alkyl chain, alkenyl chain, alkylnyl chain, a fluoroalkyl chain, branched alkyl chain, and the combination thereof.
 22. The composition as claimed in claim 20, wherein the first lipid is selected from the group consisting of: 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol), 1,2-dimyristoyl-sn-glycero-3-phosphserine, 1,2-dimyristoyl-sn-glycero-3-phosphate, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphate, 1,2-dipalmitoyl-sn-glycero-3-phosphserine, 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphate, 1,2-distearoyl-sn-glycero-3-phosphserine, 1,2-dioleoyl-3-trimethylammonium-propane, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphate, 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol), 1,2-dioleoyl-sn-glycero-3-phosphserine, 1,2-dipalmitoyl-3-trimethylammonium-propane, 1,2-distearoyl-3-trimethylammonium-propane, dimethyldioctadecylammonium bromide, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid, myristic acid, palmitic acid, stearic acid, oleic acid, tocopherols, tocotrienols, ascorbyl palmitate, sorbitan esters, glyceryl stearate, glyceryl distearates, glyceryl myristate, glyceryl palmitate, glyceryl oleate, polyoxyethylene propylene glycol stearates, and the combination thereof or derived polymers thereof.
 23. The composition as claimed in claim 20, wherein the long chain of the hydrophilic polymer moiety is selected from the group consisting of: polyethylene glycol, polypropylene glycol, polyoxyethylene, polyvinylalcohol, polyvinylpyrrolidone and related copolymers, peptide, DNA, RNA, and a combination thereof.
 24. The composition as claimed in claim 20, wherein the second lipid is selected from the group consisting of: 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000], polyoxyethylene stearates, polyethylene glycol stearates, TWEEN (Registered Trademark), Myrj™, Atlas™, d-alpha tocopheryl polyethylene glycol 1000 succinate, antibody-conjugated PEG-ylated lipid, peptide-conjugated PEG-ylated lipid, DNA-conjugated PEG-ylated lipid, RNA-conjugated PEG-ylated lipid, biotin-modified PEG-ylated lipid, maleimide-modified PEG-ylated lipid, amine-modified PEG-ylated lipid, and the combination thereof or derived polymers thereof.
 25. The composition as claimed in claim 20, wherein the one or more molecules capable of getting across a lipid membrane and decreasing van der Waals forces between lipids is selected from the group consisting of: polyethylene glycol, peptide, albumin, amino acid, sugar alcohols, butane-1,3-diol, propane-1,2,3-triol, propane-1,2-diol, propane-1,3-diol, propan-1-ol, ethane-1,2-diol, ethanol, methanol, dimethyl sulfoxide, and the combination thereof.
 26. The composition as claimed in claim 20, wherein the aqueous solvent is water, or normal saline, or buffered saline.
 27. The composition as claimed in claim 20, wherein the step of emulsifying the lipid mixture with the aqueous solvent is realized by sonication, high-speed agitation, high-pressure homogenization, or membrane filtration.
 28. The composition as claimed in claim 20, wherein the predetermined gas is selected from the group consisting of: halo-substituted hydrocarbon (perfluorocarbon), inert gas, Sulfur hexafluoride, nitrogen, oxygen, and air, or a combination thereof.
 29. The composition as claimed in claim 20, wherein the step of agitating in the mechanical manner is realized by sonication, manual shaking, high-speed mechanical agitation, microfluidic device/T-focusing, or co-axial electrohydrodynamic atomization micro-bubbling.
 30. The composition as claimed in claim 22, wherein the first lipid is selected from the group consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphate, 1,2-dipalmitoyl-sn-glycero-3-phosphserine, 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphate, 1,2-distearoyl-sn-glycero-3-phosphserine, and 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol).
 31. The composition as claimed in claim 30, wherein the first lipid is selected from the group consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, and 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol).
 32. The composition as claimed in claim 24, wherein the second lipid is selected from the group consisting of: 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000], polyoxyethylene stearates, and polyethylene glycol stearates.
 33. The composition as claimed in claim 32, wherein the second lipid is 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], polyoxyethylene stearates, or polyethylene glycol stearates.
 34. A device for producing the microbubble-containing composition of claim 20, comprising: a mechanical agitator for emulsifying the lipid mixture with an aqueous solvent to form a transparent lipid carrier solution, a temperature controlling unit for adjusting the transparent lipid carrier to 20 Celsius degree, and a sonication device, for providing agitations on the transparent lipid carrier solution; wherein the mechanical agitator includes a clamp serving to clasp the closed vessel, a lever connected to and allowed to be agitated together with the clamp, and a motor connected with the lever and providing integrally the clamp and the lever with power for reciprocation.
 35. The device as claimed in claim 34, wherein the mechanical agitator includes: a clamp, for clasping the closed vessel containing the lipid mixture dissolved in distilled water; a lever, connected with the clamp to construct a agitating mechanism; a fastener, connected to an end of the lever for restraining the movement of the lever; and a motor, connected with the lever and providing power for a reciprocation motion.
 36. The device as claimed in claim 34, further comprising a casing for providing a closed chamber and accommodating the temperature controlling unit, the mechanical agitator, and the closed vessel containing the lipid mixture, and the temperature controlling unit further comprising: a first fan set, for generating an inlet airflow flowing towards the interior of the casing; a second fan set, generating an outlet airflow flowing towards the exterior of the casing; a heating coil, heating the inlet airflow; and a sensor, for detecting a temperature within the casing so as to control the first and the second fan sets; wherein the first fan set and the heating coil are started when the temperature within the casing is to be increased to a target temperature, and the second fan set is started when the temperature within the casing is to be decreased to another target temperature.
 37. The device as claimed in claim 36, wherein the sensor is incorporated in the clamp or the lever for detect a temperature within the casing.
 38. The device as claimed in claim 34, wherein the temperature controlling unit further comprises a dry heating piece incorporated in the clamp, or the lever, or the casing for increasing the temperature within the casing. 